Apparatus and methods for cell culture

ABSTRACT

A bioreactor ( 1 ) for the culture of cells (C) comprising a stack of carriers ( 7 ) for cell (C) adherence and liquid medium (M) distribution. The carriers ( 7 ) are stacked so as to define levels ( 6 ) between adjacent carriers ( 7 ) for the flow of the liquid medium (M). Adjacent levels ( 6 ) are fluidly interconnected via open spaces ( 2 ) so that the liquid medium (M) can flow from one level ( 6 ) to an adjacent level ( 6 ). The open spaces ( 2 ) between a first and an adjacent second level ( 6 ′) do not overlap with the one or more open spaces ( 2 ) between the second level ( 6 ′) and an adjacent third level ( 6 ″). One or more of the carriers may also include an area adapted to prevent cell adhesion or growth, thereby allowing for the viewing of cell growth on adjacent carriers from a vantage point external to the bioreactor. Related methods are also disclosed.

FIELD OF THE INVENTION

The disclosure relates to an apparatus for cell culture, to a method ofmanufacture and operation of the same and to uses of the device for theculture of adherent cells, for the culture of stem cells or primarycells, for the culture of mammalian cells, and for the production ofantibodies or viruses, such as for human and/or animal therapies orvaccines.

BACKGROUND OF THE INVENTION

Cell culture of eukaryotic cells that is commercially relevant can bedivided into two classes: the cell lines and the primary or stem cells.The cell lines are relevant for the preparation of vaccines (viruses)and of proteins, e.g., antibodies. The cell is used as a bioreactor, thecell is thus nothing else than a host. It can be genetically engineered:one introduces into the cell a gene sequence whose gene product onedesires to be produced by the cells. This class of cells can be grown ina fixed bed bioreactor such as disclosed in WO 2007/039600A, thedisclosure of which is incorporated herein by reference. When dealingwith stem cells, the cells themselves can be harvested and areaccordingly the product. The product can be used, for example, forregenerative medicine and for tissue engineering.

Experiments have shown that the culture of certain types of cells suchas stem cells is much more delicate than the culture of cell lines, dueto several factors. The cells turn out to be very sensitive tomechanical stresses and other external influences. Typical procedures inuse in the production of cell lines both to expand and to harvest cellsout from a bioreactor are often not appropriate for stem cells; theyeasily damage or kill all the stem cells.

Currently, stem cells are typically grown in stationary conditions, intissues culture flasks, put in an incubator. For sake of clarity,“culture flasks” refers to all stationary culture devices, as T-Flasks,Petri dishes, Cell Factories Cell stacks and so on. Roller bottles arealso associated with culture flasks. These stationary flasks areprovided with a filter for gas exchange. The incubator comprises aplurality of T-flasks, each one constituting a substrate suitable forcell culture upon provision of adequate nutrition. Such systems have thedisadvantage of being highly inefficient in terms of the availablesurface per unit volume. Moreover, the preparation of cell growth takestime: filling T-flasks occurs by inserting a dedicated liquid (e.g.,dispersion, suspension or the like) comprising cells to be grown andnutrition, and thereafter turning the T-flask upside down once or morethan once to distribute the cells over the available levels. Inaddition, this operation needs to be repeated several times for eachbatch/each patient, based on the number of T-flasks requested to producethe sufficient amount of cells needed for a treatment.

Accordingly, a need is identified for an apparatus that addresses thelimitations of such devices and others.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide analternative bioreactor. Advantages of embodiments of the presentinvention include that they allow more cost effective culture of cells,particularly of stem cells and/or primary cells, while neverthelessallowing cells to be attached to a surface, and/or provide good growthin two dimensions and/or allow harvesting of the cells with a desiredquality level.

The object of the present invention is met by embodiments of a firstaspect of the present invention. In the first aspect of the presentinvention, a bioreactor for the culture of cells is provided.

In an embodiment of the first aspect, the disclosure relates to anapparatus for the culture of cells. The apparatus comprises a pluralityof carriers for cell adherence and liquid medium distribution. Thecarriers are stacked to define levels between adjacent carriers for theflow of liquid medium. The levels are fluidly interconnected via one ormore open spaces in the carriers so that the liquid medium can flow fromone level to an adjacent level. The one or more open spaces between afirst and a second level adjacent to the first level do not overlap witheach other.

In an embodiment of the first aspect, the disclosure relates to abioreactor for the culture of cells, comprising a stack of carriers inthe form of plates or membranes having surfaces for cell adherence andalong which liquid medium can be distributed. The carriers define levelsbetween adjacent surfaces for the flow of liquid medium, wherein thelevels are interconnected via one or more open spaces in each of thesurfaces so that liquid medium can flow from one level to an adjacentlevel. The one or more open spaces in a first carrier do not overlapwith the one or more open spaces in an adjacent second carrier whenprojected perpendicularly upon the second carrier. In other words, afirst open space in a first and second carrier on opposite side of asingle level are mutually laterally or rotationally displaced, when thefirst open space in the first carrier is seen in a perpendicularprojection upon the second open space in the second carrier. The levelsdefined between adjacent carriers are preferably hermetically closed atthe side edges of the carriers. This forces the liquid medium to use theopen spaces within a carrier to go from one level to an adjacent level.

The top and/or the bottom carrier of the stack, although not beingcommon to adjacent levels, may nevertheless possess any of thecharacteristic of any embodiment of the first aspect of the disclosure.For instance, these carriers may possess a plurality of open spaces inso that liquid medium can flow across the top or bottom carrier (toeither enter or exit the stack). The adjacent carrier in the case of abottom carrier is situated above it while the adjacent carrier in thecase of the top carrier is situated below it. In the case of any othercarrier than the top or the bottom one, an adjacent carrier is thecarrier below or above. Preferably, it is the next carrier in thedirection of the flow imposed by the driving means (and, mostpreferably, the carrier above).

In an embodiment, at least one of the carriers may be provided by singlesolid carrier (e.g., a plate or a membrane) wherein the one or more openspaces are apertures in the solid carrier. The carriers are preferablyprovided by plates or membranes. The plates or membranes can benon-porous or porous (with pores smaller than 0.05 mm so that the flowthrough the material is dominated by flow through the open spaces (e.g.circular apertures) rather than through the pores), and preferablycomprise rigid plates. Optionally, the carriers may be non-porous atleast in the area away from the interconnecting open spaces.

Alternatively, macroporous materials may be used provided the flowthrough the material is dominated by flow through the apertures ratherthan through the pores. For example, impermeable textiles can be used.Plastic materials can also be used, such as rigid polystyrene plates.

In an embodiment, at least one of the carriers may be provided by a setof solid carriers (e.g., plates and/or membranes) laterally separated bythe one or more open spaces. In the case where a set of solid carriersare laterally separated by one or more open spaces, the solid carriersare either all in the same plane (if the carrier is perpendicular to theprincipal direction), or are all part of the same conical or pyramidalsurface (if the carrier is oriented in a direction non-perpendicular tothe principal direction). The open spaces in the carrier serve as fluidinterconnects between adjacent levels. In an embodiment, the ratiobetween the overall surface area covered by the open spaces and theoverall area covered by the solid carrier(s) in each carrier may rangefrom 1% to 20%, preferably 1% to 15% and more preferably 1% to 10%. Theportion of the carrier area represented by this overall surface area ispreferably kept as small as possible so that the surface available forcarrying the cells remains as large as possible. However, a flow ofliquid medium high enough and homogeneous enough for assuring a goodoxygenation and nutrient supply for every cell requires this portion ofthe carrier area to be large enough. Embodiments of the disclosure maymeet both the cell carrier area requirement and the oxygenation/nutrientrequirement.

In an embodiment, the open spaces may have an aspect ratio (ratio of thelength on the width) from 1 to 4, preferably from 1 to 2.

In an embodiment, the open spaces may be circular.

In an embodiment, independently of the shape of the open spaces, thewidth of the open spaces may be 0.05 mm or more, 0.1 mm or more, 0.2 mmor more, 0.5 mm or more, 2 mm or less, 1 mm or less, 0.5 mm or less.Measures above 0.05 mm are preferred to allow for the passage of thecells.

In an embodiment, independently of the size and shape of the openspaces, the number of open spaces per unit area of a laminar carrier ispreferably such that the ratio between the surface area covered by theensemble of the open spaces for fluidic interconnection and the surfacearea of the solid carriers for cell adherence is preferably 20% or less,more preferably 15% or less and most preferably 10% or less. Preferably,it is 1% or more.

In an embodiment, the number of the open spaces per unit area of alaminar carrier may be constant on the whole surface of said laminarcarriers. This density of open spaces (holes) may be for instance from0.001 to 100 open spaces/mm², from 0.03 to 60 open spaces/mm² or from0.1 to 10 open spaces/mm².

In one embodiment, a plurality of open spaces is present in a firstlaminar carrier and each of these open spaces are embodied as circularholes through the laminar carrier (e.g. plate or membrane) having adiameter that is less than 1/10^(th) of the diameter of the firstlaminar carrier, preferably less than 1/50th, more preferably less than1/100^(th), most preferably less than 1/1000^(th). In this embodiment,the number of open spaces per plate may for instance be from 2 openspaces to 4*10⁷ open spaces, from 10 open spaces to 10⁷ open spaces,from 100 open spaces to 10⁶ open spaces, from 1000 open spaces to 10⁵open spaces or from 5000 open spaces to 20000 open spaces. The number ofopen spaces is of course highly dependent on the size of the laminarcarrier. In this embodiment, the number of open spaces per laminarcarrier is then suitably higher than 25, preferably higher than 50, morepreferably at least 100 and most preferably at least 1000. The diameterof the open spaces is preferably from 0.05 mm to 2 mm, more preferablyfrom 0.1 mm to 1 mm, still more preferably from 0.2 mm to 0.5 mm. Thisallows for the passage of cells.

In another embodiment, the open spaces are groove-shaped. The number ofgroove-shaped open spaces per carrier is preferably from 1 to 30, morepreferably from 2 to 20, more preferably from 4 to 25, still morepreferably from 8 to 20 and even more preferably from 12 to 18 (e.g.,16). If a number n of groove-shaped open spaces per carrier are present,two adjacent carriers have their open spaces mutually displaced by arotation of ½n turn.

The width of the grooves is preferably from 0.05 mm to 2 mm, morepreferably from 0.05 mm to 0.5 mm, even more preferably from 0.05 mm to0.1 mm to allow for the passage of cells. In the case of grooveswidening from the centre to the edge of the laminar carrier, the largerportion of the grooves has preferably a width of from 0.05 mm to 1 mm,more preferably from 0.05 mm to 0.5 mm, even more preferably from 0.05mm to 0.1 mm

In an embodiment, independently of the shape of the open spaces, thesmallest dimension of the open spaces may be 0.05 mm, preferably 0.1 mm,more preferably 0.5 mm, most preferably 1 mm.

In an embodiment, the open spaces have a shape and dimensions allowingthe passage of a spherical object of 0.05 mm diameter, preferably 0.1 mmdiameter, more preferably 0.5 mm diameter, most preferably of 1 mmdiameter.

Preferably, such groove shaped open spaces extend over a major portionbetween the center and an edge of the major surface (e.g., a majorportion of a radius in the case of a circular carrier) of a plate ormembrane. Referring to FIG. 15 (wherein the carrier comprises a set ofsolid carriers laterally separated by open spaces), an embodiment isshown wherein groove shaped open spaces extend over the whole portionbetween a center and an edge of the carrier. Grooves are advantageous asthey are more efficient in distributing the liquid medium equally tosubstantially all points of a carrier. Circular holes, due to theirdiscontinued nature, distribute the flow of liquid less homogeneously.In embodiments, the groove shaped open spaces may be subdivided into aseries of groove-shaped trenches, which improves rigidity of thecarrier.

For instance, ridges can be present on a carrier, either bridgingcarriers separated by grooves or part of a same carrier separated bygrooves. Such bridging ridges improve the rigidity of the carrier andtherefore of the device as a whole. Such bridging ridges are preferablynot penetrating the open spaces. Moreover, the architecture of thebioreactor of the disclosure preferably does not frustrate harvesting ofthe cells. In accordance with an embodiment of the invention, the openspaces are designed so as to be appropriate for flow of medium andcells. In other words, the open spaces are also intended for use duringharvesting and not merely for circulation purposes.

In an embodiment, the minimum dimension of the open spaces is 0.05 mm,preferably 0.1 mm, more preferably 0.5 mm and most preferably 1 mm. Thispermits passage of cells without damaging them.

In an embodiment, at least one of the open spaces becomes wider withincreasing radial distance from the geometrical center of the carrier.This is especially advantageous in the case of grooves. This helpsequalizing of the flow rate on the whole carrier surface.

In an embodiment, the open spaces in a carrier may be locatedrotationally symmetrically around the center. This is advantageous as itprovides a relatively homogeneous distribution of the medium over theindividual levels. Moreover, if the shape of the carriers is circular,stacking of the carriers is simplified as a rotation of a level in thestack never leads to a carrier edge standing out of the stack. This isnot the case with other carrier shapes such as oval or rectangularshapes. Such other shapes are however not excluded.

In embodiments, the surface area of the carrier (including the surfacecovered by the open spaces in the carrier) may be from 50 cm² to 1 m²,preferably from 75 cm² to 0.8 m².

For instance, in embodiments wherein the carriers are circular, theirdiameter may be from 10 to 100 cm.

The thickness of the carrier is preferably as thin as possible forallowing a higher number of carriers in a volume as small as possible.For instance, carriers having a thickness of from 0.4 to 2 mm or from0.6 to 1 mm can be used.

In an embodiment, the overall surface area covered by the open spaces inat least one of carrier increases faster with increasing radial distanceto the geometrical center of the carrier. This is advantageous becauseit reduces the difference in flow rate experienced by the cells close tothe geometrical center of the carrier and by the cells farther away fromthe geometrical center of the carrier. This allows optimization so thatthe flow rate can be made substantially equal at every distance of thegeometrical center of the carrier. Preferably, the open spaces have asurface area and are designed and/or distributed such that the overallsurface area of open spaces increases with increasing radial distance toa center. This turns out to improve homogeneity of the flow rate acrossthe bioreactor. It is observed for clarity that this increased overallsurface area may be obtained by increase of the width of single openspaces. Alternatively or additionally, it may be obtained by increase ofthe number of open spaces. The increase in surface area may becontinuous or could be discontinuous, e.g., stepwise. In case that thebioreactor has a cylindrical shape, which is preferred, the increase ofoverall surface area will be independent of the location or phase of theopen spaces. In case that the bioreactor has another shape, for instancewith an oval or rectangular cross-section perpendicular to the principaldirection, the increase in surface area may be different for differentdirections away from the center.

In an embodiment, the overall surface area covered by the open spaces inat least one of the plates or membranes increases faster with increasingradial distance to the geometrical center of the carrier in such a waythat the flow rate is substantially equal at every distance of thegeometrical center of the carrier.

In all embodiments, the one or more open spaces in a first carrier donot overlap with the one or more open spaces in an adjacent secondcarrier when projected perpendicularly upon the second carrier. This isadvantageous because this prevents the liquid medium from travelling ina straight line between adjacent levels and forces at least some of theliquid medium to flow laterally within a level before being able toreach an adjacent level (typically the adjacent upper level). Althoughthe global flow of the liquid medium is along the principal direction(e.g., the liquid may globally travel from the bottom of the stack ofcarriers up to the top of the stack), the liquid medium spends more timeflowing (e.g., laterally) within a level than across (e.g., vertically)adjacent levels. This enables the liquid medium to reach all area ofeach surface of a carrier and permits therefore to have substantiallythe same flow everywhere in the bioreactor.

In other words, first open spaces in a first and second carrier onopposite side of a single level are mutually rotationally or laterallydisplaced, when the first open space in the first carrier is seen in aperpendicular projection upon the second carrier. With such a rotationalor lateral displacement, a risk is avoided that a set of open spacesdefines a columnar channel extending over several levels. In animplementation thereof, the at least one open space in the first carrieris provided with a first phase between a lower and an upper angle, thephase being defined as a phase or orientation around a center of thelevel. The open space in the second carrier is provided with a secondphase between a lower and an upper angle, wherein the first phase doesnot overlap with the second phase.

An embodiment of the first aspect relates to a bioreactor for theculture of cells, comprising a stack of laminar carriers for celladherence and liquid medium distribution, said laminar carriers beingstacked in a principal direction, each pair of adjacent laminar carrierswithin said stack being separated from one another by an averagedistance so as to define a level between said adjacent laminar carriersfor the flow of said liquid medium, wherein adjacent levels are fluidlyinterconnected via a plurality of open spaces in a laminar carriercommon to said adjacent levels so that said liquid medium can flowbetween said adjacent levels, wherein said open spaces in a laminarcarrier have a width at least five times and preferably at least tentimes smaller than the average distance separating said laminar carrierfrom an adjacent laminar carrier, and wherein said width is at least0.05 mm. It has been surprisingly observed that when the open spaceshave a width at least five times and preferably at least ten timessmaller than said average distance, even in the case when the openspaces of a first laminar carrier overlap when projected perpendicularlyupon the open spaces of a second laminar carrier, the liquid medium donot travel in a straight line between adjacent levels and at least someof the liquid medium flow laterally within a level before being able toreach an adjacent level (typically the adjacent upper level). Althoughthe global flow of the liquid medium is along the principal direction(e.g. the liquid may globally travel from the bottom of the stack oflaminar carriers up to the top of the stack of laminar carriers), theliquid medium spends more time flowing (e.g. laterally) within a levelthan across (e.g. vertically) adjacent levels. This enables the liquidmedium to reach all area of each surface of a laminar carrier andpermits therefore to have substantially the same flow everywhere in thebioreactor.

In an embodiment wherein said open spaces in a laminar carrier have awidth at least five times and preferably at least ten times smaller thanthe average distance separating said laminar carrier from an adjacentlaminar carrier, and wherein said width is at least 0.05 mm, the openspaces may have an aspect ratio (ratio of the length to width) from 1 to4, preferably from 1 to 2.

In an embodiment wherein said open spaces in a laminar carrier have awidth at least five times and preferably at least ten times smaller thanthe average distance separating said laminar carrier from an adjacentlaminar carrier, and wherein said width is at least 0.05 mm, the openspaces may be circular.

In an embodiment, wherein said open spaces in a laminar carrier have awidth at least five times and preferably at least ten times smaller thanthe average distance separating said laminar carrier from an adjacentlaminar carrier, and wherein said width is at least 0.05 mm,independently of the shape of the open spaces, the width of the openspaces may be 0.05 mm or more, 0.1 mm or more, 0.2 mm or more, 0.5 mm ormore, 2 mm or less, 1 mm or less, 0.5 mm or less. Measures above 0.05 mmare preferred to allow for the passage of the cells.

In an embodiment wherein said open spaces in a laminar carrier have awidth at least five times and preferably at least ten times smaller thanthe average distance separating said laminar carrier from an adjacentlaminar carrier, and wherein said width is at least 0.05 mm.

It must be noted that the top and/or the bottom laminar carrier of thestack, although not being common to adjacent levels, may neverthelesspossess any of the characteristic of any embodiment of the first aspectof the present invention. For instance they may possess a plurality ofopen spaces in so that said liquid medium can flow across said top orbottom laminar carrier (to either enter or exit the stack of layer),wherein said open spaces in a laminar carrier have a width at least fivetimes and preferably at least ten times smaller than the averagedistance separating said laminar carrier from an adjacent laminarcarrier, and wherein said width is at least 0.05 mm. The adjacentlaminar carrier in the case of a bottom laminar carrier is situatedabove it while the adjacent laminar carrier in the case of the toplaminar carrier is situated below it. In the case of any other laminarcarrier than the top or the bottom one, an adjacent laminar carrier isthe laminar carrier below or above. Preferably, it is the next laminarcarrier in the direction of the flow imposed by the driving means.Preferably, it is the laminar carrier above.

Within the stack of carriers, the laminar carriers are preferablyparallel to each other. In this case, the average distance is a fixeddistance.

The average or fixed distance between two laminar carriers is preferablyfrom 0.5 to 10 mm, preferably from 1 to 5 mm, more preferably from 1 to3 mm and even more preferably from 1.2 to 2 mm. The present embodimenthas the advantage of viably (for the cells) enabling such a smallinter-level distance. In static cell culture devices, such a smallinter-level distance would not permit enough space above the cells topermit sufficient oxygenation of the cells. Also, the presence of aplurality of open spaces in the laminar carriers, wherein said openspaces have a width at least five times and preferably at least tentimes smaller than said average distance enable such small inter-leveldistances to be used by decreasing the time necessary for the liquidmedium to travel from the bottom to the top of the stack of laminarcarriers while simultaneously decreasing the liquid flow experienced bythe cells.

In a further embodiment, the minimum dimension of the open spaces is atleast a tenth and preferably at least a fifth of the distance betweentwo carriers.

In a further embodiment wherein said open spaces in a laminar carrierhave a width at least five times and preferably at least ten timessmaller than the average distance separating said laminar carrier froman adjacent laminar carrier, and wherein said width is at least 0.05 mm,the minimum dimension of the open spaces is at least a tenth andpreferably at least a fifth of the distance between two laminarcarriers.

In a further embodiment, at least some of the carriers have at least oneside edge provided with at least one ridge, ridges of first and secondadjacent carriers defining a mutual distance between the carriers at theside edge. This arrangement provides structural stability. The ridge mayextend on a first (e.g., top) side and/or on a second (e.g., bottom)side of the side edge. The ridges may be continuous along the side edge.Alternatively, the ridges may be block-shaped and mutually separated byspaces. Block-shaped ridges of an adjacent carrier may extend into suchspaces. This allows the definition of a mutually fixed position. Inother words, female and male notches for plate positioning may bepresent at the edge of each carrier. Ridges may also project outwardfrom the edges.

In an embodiment, the levels defined between adjacent carriers areclosed at the side edges of the carriers. The closure of the side edgesof the carriers will result in defining the side outer surface of thebioreactor. The stacking occurs in one embodiment by means of mechanicalconnections defining the side wall of the columnar channel. Thesemechanical connections may fix the orientation of each carrier in thestack, but alternatively may leave freedom for independent rotation ofeach of the carriers. Clearly, it is by no means excluded that the stackof carriers including the columnar channel is manufactured as one piece,for instance by means of a moulding process, and/or that adhesive ormechanical fixtures (screw or the like) are used for fixing portion ofthe stack. However, separate manufacture of the carriers has theadvantage that the stack becomes modular, so as to be made larger orshorter dependent upon the intended use and needed conditions.

Preferably use is made of the ridges for providing a closure. Theclosures of the levels may be obtained by connecting ridges (e.g.,ridges projecting outward from the edges) with a polymer material. Suchpolymer materials include adhesives, resins and the like. The polymermaterial may be applied as a liquid or sheet like material and then beprocessed, for instance dried, cross-linked or may be first melted andthen allowed to cool down and solidify. The polymer material mayalternatively be moulded into a desired shape, for instance byinsert-moulding or transfer moulding.

The stack of carriers may comprise from 5 to 500 carriers defining from5 to 500 levels. Since two adjacent carriers define one level, thenumber of levels is normally the number of carriers minus one. However,the top carrier of the stack is usually also usable for the culture ofcells which means that the number of carriers can be equal to the numberof levels.

The number of levels and/or carriers within one bioreactor is forinstance in the range of 5 to 500, preferably in the range of 80 to 200and more preferably in the range of 130 to 180.

Preferably, all carriers are structurally the same, i.e., have the samedimensions, the same orientation, the same area ratio openspaces/carrier and have open spaces having the same shapes anddimensions. This allows for the same flow rate at each level.Preferably, the average or fixed distance is the same for each pair oflaminar carrier in the stack of laminar carrier. This also permits tohave the same flow rate at each level. Such equal flow rate (within alevel and/or between level) may be desired, even if not essential, inorder to guarantee that same culture conditions apply everywhere insidethe bioreactor.

With the relatively constant distribution across each carrier andprimarily lateral flow direction, the cells may be attached to thecarrier and a good cell growth based on a two-dimensional culture may beobtained. The open spaces in a carrier may be suitably embodied asapertures in the form of holes or grooves.

In a preferred embodiment of the disclosure, each carrier of the stackcomprises at least two open spaces. In such a case, the bioreactor ofthe disclosure can be considered to be composed of a plurality of fluidinterconnects that are coupled both in series and in parallel. Thiscombination of series and parallel coupling of fluid interconnects—whichare effectively portions of a level—turns out better than either aseries coupling or a parallel coupling of fluid interconnects alone. Ifthe fluid interconnects were coupled merely in series, they would extendmerely laterally on one level. In particular, the medium would flow on afirst level from a first aperture to a distant second aperture and thenback on a subsequent second level and so on. When each carrier of thestack comprises at least two open spaces, the flow rate can be lowerthan if only one open space per carrier is present while keeping thesame circulation time. The shear stress can therefore be made lower orthe circulation time can be reduced. In the case of only one open spaceper carrier the circulation time can be very high, e.g., easily a coupleof hours, with a flow rate that limits shear stress (for instance equalto or smaller than 2 mm/s).

Useful embodiments when only one open space is present per plate are forinstance embodiments where an external circulation system is used andwherein alternating carriers in the stack have alternatively a centralopen space and a peripheral open space. The carriers in this embodimentare of two alternative kinds. For instance, a first kind is hold inplace by its edge attached an external wall and has a central openspace. In this example, a second kind is hold in place by being attachedto a central axis and assures an open space for fluidly interconnectingtwo adjacent levels by not extending to the wall closing the stack. Ofcourse, the central axis can be a fluid channel (e.g., a hollow tube)concentric and internal to the stack of carriers. This hollow tube formsa fluid channel separate from the stack of carriers providing a fluidconnection between a first carrier at a first extremity of the stack ofcarriers and a second carrier at a second extremity of the stack ofcarriers, therewith providing a circulation system for the liquidmedium.

Alternatively, if the fluid interconnects were merely coupled inparallel, one fluid interconnects would likely be shorter than anotherone. Hence pressure drop would be inhomogeneous within the bioreactor,leading to differences in flow rate within the system. Such majordifferences in flow rate are undesired, as they may lead to turbulenceand/or inhomogeneities that may easily damage cells and cell growth.Moreover, flow rate in some fluid interconnects of the bioreactor may below. This will typically lead to dead zones (zones with poor mixing)and, consequently, differences in cell growth over the bioreactor. In aworst case scenario, cell culture in certain fluid interconnects wouldnot lead to an adequate final product of the desired quality.

Moreover, the serial and parallel coupling of fluid interconnectsprevents that a single obstruction of a fluid interconnects (e.g., anopen space or aperture) blocks substantially all further flow.

In an embodiment, the reactor is provided with a first and an oppositesecond side (e.g., a bottom and a top) and comprises a stack of carriersas defined in any embodiment of the first aspect of the disclosure,e.g., that are stacked in a principal direction from the first side(e.g., the bottom of the reactor) to the second side (e.g., the top ofthe reactor), so as to define levels between adjacent carriers for theculture of the cells and flow of medium. It will be understood by theskilled person that the bioreactor is at its second side (e.g., top)preferably closed so as to maintain physical conditions in the bestmanner. In embodiments, the top and/or bottom of the reactor maycomprise inlet and/or outlet (e.g., an inlet and outlet for gas) and/orprobes (e.g., a temperature probe, pH probe, dissolved O₂ probe,dissolved CO₂ probe, biomass probe or any other probe). The top and/orbottom may also be at least partly transparent to allow microscopemediated observation. For instance, the first and/or second side maycomprise optically transparent windows.

In an embodiment, the reactor may comprise at least one probe formeasuring a parameter of the liquid medium (e.g., a physical, chemicalor biological parameter such as the temperature, the pH, the O₂concentration, the CO₂ concentration, the cell density in the medium(biomass), among others). The bioreactor may further comprise acontroller connected to the probe for modifying the parameter infunction of the input received by the controller from the probe. Inembodiments, the bioreactor may further comprise means for modifying theparameter. In embodiments, the means are connected to the controller.Examples of such means are heating means, cooling means, gas deliverymeans and driving means amongst others.

In another embodiment, the bioreactor further is provided with a pump ormedium circulation means (e.g., an impeller) for circulation of themedium in the principal direction. The advantages are among othersbetter aeration, heat transfer and possibility of control. Furthermore,in accordance with the disclosure, fluid circulation is enabled suchthat medium can be circulated also during harvesting.

In this manner, ⁻the bioreactor of the invention provides atwo-dimensional structure for cell culture with appropriate medium flowand harvesting.

The risk of generating an entirely vertical flow of the medium overseveral levels that leads to an inhomogeneous distribution of cells andmedium, which can be reduced by having the width of the open spaces atleast five times smaller than the distance between two laminar carriersor which can be reduced by having open spaces not overlapping betweenadjacent laminar carriers, may be is alternatively or additionallyreduced by means of the shape of the fluid interconnects and/or throughactive flow stimulation. The latter active flow stimulation is suitablyarranged through a circulation means (e.g., an impeller such as amechanical impeller and preferably a magnetic impeller) as known in theart. The impeller is suitably provided in an upper cavity above thestack of laminar carriers and adjacent to it or in a lower cavity at abottom of the bioreactor (under the stack of laminar carriers) andadjacent to it. Alternatively, a first impeller is present in the lowercavity and a second impeller is present in the upper cavity, so as toallow operating the bioreactor upside down. Preferably, bearings areprovided in the lower and/or upper cavity for positioning said impeller.The impeller can be present in the fluid channel (e.g., in a central andconcentric column providing a fluid connection between said firstlaminar carrier and said second laminar carrier and providing acirculation system for the liquid medium) This is especiallyadvantageous when no lower cavity is present.

In an embodiment of the disclosure, the bioreactor may further comprisea top zone and a bottom zone adjacent to respectively the first and thesecond extremity of the stack of carriers and in fluid communicationtherewith and with the central fluid channel.

In an embodiment of the disclosure, the bioreactor may further compriseinlet/outlet ports to the top and/or bottom zone.

In an embodiment of the disclosure, at least one inlet port may bepresent in the bottom zone and at least one outlet port may be presentin the top zone and external circulation means such as a pump may becoupled between the inlet and outlet ports.

In again another embodiment, at least some of the carriers have aportion that is oriented in a direction including a non-perpendicularangle to the principal direction, which portion comprises at least oneopen space. A preferred implementation hereof is that the carriers havea curved shape, when seen in a cross-sectional side view through acenter axis. For instance, the carrier may be conical. Alternatively,the carrier can also be pyramidal. Though it is deemed suitable thatsubstantially all carriers have the same or a similar shape andorientation, this is not deemed necessary. For instance, a first carriermay be of curved (e.g., conical or pyramidal) shape with an edge that isnearer to the first side of the bioreactor than its center, while asecond carrier is of curved shape and is provided with an edge that isnearer to the second side of the bioreactor that its center—in otherwords, the orientation may be opposite. It may further be that the firstcarrier is flat and the second is of a different shape (e.g., conical).It may further be that the first and the second carriers are curved andhave same orientation, but that the curvature of the first carrierdiffers from that of the second carrier. Such embodiments lead thereto,that a level has varying height, which may be advantageous for improvedharvesting. Additionally, a stack may be subdivided into a first and asecond substack, between which an inlet and/or outlet for fluid and/ormedium may be present. Such port is then suitably coupled to a levelwith a varying height.

In one further embodiment, physical conditions in the bioreactor may bemonitored and controlled adequately. This is deemed beneficial as stemcells/primary cells are very fragile. Slight variations in physicalconditions such as temperature, biomass, pH, O₂ and CO₂-concentrationsand mechanical shocks may damage the stem cells. The bioreactor of thedisclosure is thereto suitably provided with an upper cavity on top ofthe stack of carriers and adjacent to the stack of carriers. Sensors maybe present at least in the upper cavity.

Sensors may be present inside the bioreactor. In addition oralternatively, composition and physical conditions of the externalcirculation system, if any, may be monitored, for instance between theoutlet port of the bioreactor and the medium storage tank. A separatesensing vessel may be foreseen for this. Typical conditions to bemeasured include the pH, the temperature, the biomass, the oxygen andCO₂ content of the medium, the amount of biological material, and/or theeffective flow rate. The bioreactor according to the disclosure maypreferably comprise at least one fluid channel separate from the stackof carriers providing a fluid connection substantially extending fromthe first side to the second side, therewith providing an internalcirculation system of medium. An internal circulation system for mediumleads to smaller footprint than an external circulation system.Moreover, the number of external components is less than with anexternal circulation system, which is beneficial for user friendliness.Expressed in other words, in an embodiment, the bioreactor may furthercomprise at least one fluid channel separate from the stack of carriersproviding a fluid connection between a first carrier at a firstextremity of the stack of carriers and a second carrier at a secondextremity of the stack of carriers, therewith providing a circulationsystem for the liquid medium. Preferably, the fluid channel is embodiedas a columnar channel that is located on a center axis of thebioreactor. In an embodiment, the fluid channel may be concentric to thestack of carriers and internal or external to the stack of carriers. Forinstance, the fluid channel may be a central column internal to thestack of carriers.

The columnar channel suitably extends between a lower and an uppercavity. An impeller may be present in the lower cavity and/or in theupper cavity. The impeller has preferably its rotational axis confoundedwith the center axis. As mentioned above, the impeller can be in thecolumnar channel e.g., if no lower cavity is present. In this case, atleast some ports need to be present to interconnect the columnar channelwith at least one level (preferably comprising the lowest level of thestack). It is observed that the lower cavity may be separated from thecolumnar channel by means of a wall having one or more apertures, i.e.,access ports for the medium. Such separation allows that the flow in thecolumnar channel is more vigorous than in the levels of the reactor. Insuch a manner both an appropriate mixing and an appropriate flow ratefor the cells may be achieved. Ports between such columnar channel andindividual levels of the bioreactor may be present but are preferablynot present. However, as mentioned above, if no bottom cavity is presentand if the circulation means or pump is an impeller in the columnarchannel, port(s) are preferably present. Suitably, the columnar channelis not provided with such ports, but with means, such as a tube or thelike, for providing components into medium within the columnar channel.Examples of such components are gaseous components such as air, oxygenand CO₂, but also acid or base to correct pH, or culture medium, orother nutrients or additives. The means may be embodied as a tube, butcould alternatively be embodied as means for addition of components insolid form. If the means are embodied as a tube for gas regulationand/or exchange and/or control and regulation of gas (such as oxygen orcarbon dioxide concentration), the means could be a simple tube forinfecting gas bubbles or could be a closed porous silicon tubing (thisavoids bubbles and foam). The columnar channel may then have quite somestirring or at least rotational movement. As a result of which thecomponents, such as oxygen, may dissolve into the medium, and anappropriate mixing is achieved prior to provision of the medium to thelevels where cells are growing. Furthermore, any bubbles may leave themedium prior to its provision to the individual levels. Bubbles goingthrough the levels might damage cells and are thus undesired.

Such means for providing components into the medium can also be providedat other locations (e.g., in the upper cavity). A filter for gasexchange can also be provided.

In again a further embodiment, the level comprises a structured fixedbed with cavity sizes adapted to the size of the cells to be expanded.Moreover, harvesting may be carried out without shaking the bioreactor.Such shaking has the disadvantage of likely destroying grown cells, incase of culture of stem cells or primary cells.

In an embodiment, the carriers of the bioreactor are hydrophilic. Thiscan be achieved via a surface treatment. Surface treatment isadvantageous for cell cultivation. Basically, hydrophilisation issuitably carried out by a physical (e.g., vacuum or atmospheric plasmatreatment) or a chemical treatment (functionalisation with hydrophilicsilanes). Other treatments, more sophisticated, can be envisaged(physical, chemical functionalization for example).

Harvesting may be carried out, such as by an enzymatic reaction (e.g.,as with trypsine). Anchoring points on the surface of the carrier may bepresent for adhesion, though a smooth surface is typically preferred.

In an embodiment, the bioreactor of the disclosure is adapted so thatthe linear velocity of the medium in the levels is lower than 2 mm/s(e.g., 1 mm/s) and/or so that the circulation time of the medium islower than 60 minutes (e.g., 30 min). This can be achieved by selectingappropriate level width, open space density, impeller or pumping speedand open space shape and location.

Preferably, the bioreactor is further adapted to permit the linearvelocity of the medium to be increased to the range 10 mm/s-20 mm/s forthe harvesting step. This helps in detaching the cells from thecarriers.

The bioreactor according to embodiments of the disclosure is mostsuitably used for the culture of animal cells (e.g., mammalian cells,insect cells, fish cells, plant cells or the like), preferably mammaliancells including or not including human cells but preferably includinghuman cells. The bioreactor according to embodiments of the disclosurecan be used for the culture of primary cells and/or stem cells. However,it is not excluded that the bioreactor according to the invention findsapplication for culture of any other type of cell or even any other typeof biological material, such as viruses, bacteria and the like.

In a second aspect, the disclosure relates to a method of cultivatingcells. In an embodiment of this second aspect, the method comprises thesteps of:

-   -   Sensing a value of a parameter of the liquid medium (e.g., pH,        temperature, O₂ concentration, CO₂ concentration, . . . ), and    -   Circulating the liquid medium in the bioreactor by operating a        circulation means or pump as long as the value of the parameter        is below or above a predetermined value.

In a particular embodiment of this second aspect, the method comprisesthe steps of:

-   -   Sensing a value of a parameter of the liquid medium (e.g., O₂        concentration), and    -   Circulating the liquid medium in the bioreactor by operating a        circulation means or pump as long as the value of the parameter        is below a predetermined value.

In another embodiment of this second aspect, the method comprises thefollowing collecting steps once the cells are grown in a bioreactoraccording to any embodiment of the first aspect of the disclosure:

-   -   Emptying the bioreactor from its liquid (culture) medium by        opening an outlet for the liquid medium,    -   Introducing another liquid medium comprising a releasing agent        (e.g., trypsin),    -   Circulating the other liquid medium within the bioreactor by        operating circulation means or pump,    -   Emptying and thereby collecting the grown cells.

In another embodiment of the second aspect of the disclosure, the methodcomprises the steps of:

-   -   Introducing a first liquid medium comprising cells in a        bioreactor according to any embodiments of the first aspect of        the disclosure,    -   Operating circulation means or pump in order to circulate the        first liquid medium in the bioreactor,    -   Stopping the circulation means or pump in order to allow the        cells to settle on a first side of the carriers of the        bioreactor, and    -   Optionally removing the liquid medium from the bioreactor,        introducing a second liquid medium comprising cells in the        bioreactor, operating the circulation means or pump in order to        circulate the first liquid medium in the bioreactor, stopping        the circulation means or pump, turning the bioreactor upside        down and allowing the cells to settle on a second side of the        carriers of the bioreactor,

In a further embodiment of the second aspect of the disclosure, themethod comprises a combination of the steps above. For instance, themethod may comprise the steps of:

-   -   Introducing a first liquid medium comprising cells in a        bioreactor according to any embodiments of the first aspect of        the disclosure,    -   Operating circulation means or pump in order to circulate the        first liquid medium in the bioreactor,    -   Stopping the circulation means or pump in order to allow the        cells to settle on a first side of the carriers of the        bioreactor,    -   Optionally removing the liquid medium from the bioreactor,        introducing a second liquid medium comprising cells in the        bioreactor, operating the circulation means or pump in order to        circulate the first liquid medium in the bioreactor, stopping        the circulation means or pump, turning the bioreactor upside        down and allowing the cells to settle on a second side of the        carriers of the bioreactor, Sensing the value of a parameter of        the liquid medium (e.g., oxygen concentration),    -   Circulating the liquid medium in the bioreactor by operating the        circulation means or pump as long as the value of the parameter        is below a predetermined value,    -   Once the cells are grown, emptying the bioreactor from its        liquid (culture) medium by opening an outlet for the liquid        medium,    -   Introducing another liquid medium comprising a releasing agent        (e.g., trypsin),    -   Circulating the other liquid medium within the bioreactor by        operating a circulation means or pump,    -   Emptying and thereby collecting the grown cells.

Yet another aspect of the disclosure relates to apparatus and methodsfor monitoring cell growth including multiple stacked carriers. A firstcarrier positioned above a second carrier may include an area or portionthat is adapted to prevent the growth of cells. When this area orportion is aligned with a growth area of an underlying carrier, itallows a line of sight to be maintained from external to the bioreactorand through the first carrier to the growth area.

Still a further aspect of the disclosure relates to a carrier forculturing cells in a bioreactor. The carrier includes a surface havingan area adapted for cell adherence and an area adapted for preventingcell adherence. Preferably, the surface for preventing cell adherence isassociated with a piece of transparent material, or may be hydrophobic.

A bioreactor for the culture of cells may include first and secondcarriers, each having at least one inlet for admitting a fluid medium.The first carrier including a first portion adapted for cell adherence,and a second of the carriers further includes a second portion adaptedto prevent cell adherence. The second portion of the second carrier thusallows for viewing the first portion of the first carrier when the firstand second portions align.

Preferably, the first and second portions comprise surfaces on thecarriers, but may form openings as well. The second portion may includean optically transparent material between the second carrier and thefirst carrier. A single housing may be provided for receiving thecarriers, or the carriers may comprise stackable trays or cubes.

A third carrier may be provided having the first portion adapted forcell adherence and a third portion adapted for preventing cell adherencefor aligning in use with the second portion of the second carrier.

Still another aspect of the disclosure is a bioreactor for the cultureof cells. The bioreactor comprises a stack of carriers. A first of thecarriers positioned above a second of the carriers includes an areaadapted to prevent cell adherence. The area of the first carrier withoutcell adherence allows for viewing a cell growth area on the secondcarrier through the first carrier.

A bioreactor for the culture of cells comprises a stack of N carriers.Carrier N-1 has at least one area adapted to prevent cell adherence.This area of carrier N-1 allows for viewing a cell growth area oncarrier N through carrier N-1. Preferably, carrier N-2 has at least twoareas adapted to prevent cell adherence, a first of the prevent areasallowing for viewing of a first cell growth area on carrier N-1 and asecond of the prevent areas allowing for the viewing of a second cellgrowth area on carrier N using a line of sight through the prevent areaof carrier N-1.

Viewed another way, a bioreactor for the culture of cells comprises astack of N carriers. Carrier N-M has at least M area(s) adapted toprevent cell adherence. These area(s) of carrier N-M allows for viewinga cell growth area on carrier N through carrier N-M.

The disclosure also related to a bioreactor comprising a stack of Ncarriers and having an optically transparent line of sight from anexternal vantage point to a cell growth area on a surface of carrierN-1. Preferably, a column of transparent material forms the opticallytransparent line of sight.

Viewed another way, a bioreactor comprising a stack of carriers includesan optically transparent solid material positioned in a space betweenadjacent carriers.

Still a further aspect of the disclosure is a bioreactor comprising avertical stack of carriers, each having an inlet for receiving a fluidmedium, and adapted to allow for viewing a growth area on one carrierthrough an adjacent carrier other than via the inlet.

Yet another aspect is a bioreactor for the culture of cells comprising astack of carriers. The carriers are stacked so as to define levelsbetween adjacent carriers for the flow of a liquid medium from an inletto an outlet of the bioreactor. Adjacent levels are fluidlyinterconnected via two or more open spaces so that the liquid medium canflow from one level to an adjacent level. At least one of the openspaces provided by a first carrier at least partially overlaps with anopen space provided by a second, adjacent carrier to create asubstantially unobstructed optical path to view a growth area on a thirdcarrier. Preferably, the carriers further include one or more openspaces between a first and an adjacent second level that do not overlapwith the one or more open spaces between the second level and anadjacent third level when projected along the principal direction.

The disclosure also pertains to a bioreactor for the culture of cellscomprising a stack of at least three carriers. A first carrier providesat least three open spaces without cell adherence, and at least one openspace on the first carrier aligns with at least one open space on asecond carrier to allow for viewing a cell growth area on a thirdcarrier. Preferably, the second carrier comprises at least two openspaces.

BRIEF DESCRIPTION OF THE FIGURES

The bioreactor according to the invention will be further elucidatedwith reference to the figures, in which:

FIG. 1 shows a diagrammatical view of a bioreactor provided with anexternal circulation system, according to an embodiment of thedisclosure;

FIG. 2 shows a diagrammatical cross-sectional view of a bioreactorcomprising a circulation system integrated in the bioreactor accordingto an embodiment of the disclosure;

FIG. 3 shows a diagrammatical top view of a carrier according to adesign for use in a bioreactor according to an embodiment of thedisclosure;

FIG. 4 shows a diagrammatical top view of a carrier according to adesign for use in a bioreactor according to an embodiment of thedisclosure;

FIG. 5 shows a diagrammatical top view of a carrier according to adesign for use in a bioreactor according to an embodiment of thedisclosure (left); it also shows a graph showing that the overallsurface area (A) covered by the open space in the carrier increasesfaster with increasing radial distance (D) to the geometrical center ofthe carrier;

FIG. 6 shows a diagrammatical top view of a carrier according to adesign for use in a bioreactor according to an embodiment of thedisclosure;

FIG. 7 shows a diagrammatical top view of a carrier according to adesign for use in a bioreactor according to an embodiment of thedisclosure;

FIG. 8 a-8 c shows figures for explanation of the flow in a bioreactoraccording to embodiments of the disclosure;

FIGS. 9 and 10 show diagrammatical cross-sectional views of furtherembodiments of the disclosure wherein the bioreactor comprises carriersincluding a non-perpendicular angle relative to the principal directionin the bioreactor;

FIGS. 11 and 12 show diagrammatical cross-sectional views of furtherembodiments of the disclosure wherein the bioreactor comprises carriersincluding a non-perpendicular angle relative to the principal directionin the bioreactor;

FIGS. 13 a-13 a show a method according to an embodiment of the secondaspect of the disclosure;

FIG. 14 shows a diagrammatical cross-sectional view of a bioreactoraccording to an embodiment of the disclosure;

FIG. 15 shows a diagrammatical top view of a carrier according to adesign for use in a bioreactor according to an embodiment of thedisclosure;

FIG. 16 shows a perspective view of a carrier according to a design foruse in a bioreactor according to an embodiment of the disclosure;

FIG. 17 shows a diagrammatical top view of a carrier according to adesign for use in a bioreactor according to an embodiment of thedisclosure (left); it also shows a graph showing that the overallsurface area (A) covered by the open space in the carrier increasesfaster with increasing radial distance (D) to the geometrical center ofthe carrier;

FIG. 18 shows a cross-sectional view of the edge of a stack of carriersfor use in an embodiment of a reactor according to the disclosure.

FIG. 19 shows a perspective view of a stack of carrier according to anembodiment of the disclosure.

FIGS. 20-26 relate to a bioreactor according to an embodiment of thedisclosure.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosure will be described with respect to particular embodimentsand with reference to certain drawings but the invention is not limitedthereto but only by the claims. The drawings described are onlyschematic and are non-limiting. In the drawings, the size of some of theelements may be exaggerated and not drawn on scale for illustrativepurposes. The dimensions and the relative dimensions do not correspondto actual reductions to practice of the invention. It should beappreciated that in the description of exemplary embodiments of theinvention, various features of the invention are sometimes groupedtogether in a single embodiment, figure, or description thereof for thepurpose of streamlining the disclosure and aiding in the understandingof one or more of the various inventive aspects. This method ofdisclosure, however, is not to be interpreted as reflecting an intentionthat the claimed invention requires more features than are expresslyrecited in each claim. Rather, as the following claims reflect,inventive aspects lie in less than all features of a single foregoingdisclosed embodiment. Thus, the claims following the detaileddescription are hereby expressly incorporated into this detaileddescription, with each claim standing on its own as a separateembodiment of this invention.

Furthermore, while some embodiments described herein include some butnot other features included in other embodiments, combinations offeatures of different embodiments are meant to be within the scope ofthe invention, and form different embodiments, as would be understood bythose in the art. For example, in the following claims, any of theclaimed embodiments can be used in any combination.

Furthermore, some of the embodiments are described herein as a method orcombination of elements of a method that can be implemented by aprocessor of a computer system or by other means of carrying out thefunction. Thus, a processor with the necessary instructions for carryingout such a method or element of a method forms a means for carrying outthe method or element of a method. Furthermore, an element describedherein of an apparatus embodiment is an example of a means for carryingout the function performed by the element for the purpose of carryingout the invention.

In the description provided herein, numerous specific details are setforth. However, it is understood that embodiments of the invention maybe practiced without these specific details. In other instances,well-known methods, structures and techniques have not been shown indetail in order not to obscure an understanding of this description.

FIG. 1 shows a diagrammatical view of a first embodiment of thebioreactor according to the invention. The bioreactor 1 is provided witha first side 11 and an opposite second side 12. A stack of carriers 7,which are preferably, is present in the bioreactor 1. The carriers arestacked along a principal direction P extending from the first side 11to the second side 12. The carriers 7 are provided with open spaces (notshown). The bioreactor 1 of this embodiment is provided with an externalcirculation system 30. The external circulation system 30 comprises amedium storage tank 32 that is coupled to the bioreactor 1 through tubes36. An external pump 33 is present for enabling flow of medium throughthe bioreactor 1. Medium flowing through the tubes enters the bioreactor1 at inlet port 21 on the first side 11 of the bioreactor 1. It passeseach carrier 7 within the bioreactor 1 through the open spaces therein,and then leaves the bioreactor at outlet port 22 on the second side 12of the bioreactor 1. The medium storage tank 32 is in this exampleprovided with a filter 34 for gas exchange and with means or transporter35 for addition of components. The means 35 may be embodied as a tube,but could alternatively be embodied as means for addition of componentsin solid form. While the medium storage tank 32 is shown here in atypical laboratory implementation, e.g., a beaker glass, it will beclear that implementations of larger scale are not excluded. While themedium storage tank 32 is shown here to be coupled to a singlebioreactor 1, it is not excluded that it is coupled to a plurality ofbioreactors 1, suitably arranged in parallel. Though not shown, it ispreferred that the composition and physical conditions of the bioreactor1 are monitored. Hereto, sensors may be present inside the bioreactor.Alternatively, composition and physical conditions of the externalcirculation system may be monitored, for instance between the outletport 22 of the bioreactor 1 and the medium storage tank 32. A separatesensing vessel may be foreseen for this. Typical conditions to bemeasured include the pH, the temperature, the oxygen and CO₂ content ofthe medium, the amount of biological material, and/or the effective flowrate.

FIG. 2 is a cross-sectional diagrammatical view of a second embodimentof the bioreactor 1. The embodiment shown here is a bioreactor in whicha circulation system is integrated. In this example the reactor isprovided with a lower cavity 3, an upper cavity 4 and a fluid channel 5extending between the lower and upper cavity 3, 4 along the principaldirection P of the bioreactor 1. The fluid channel 5 is in the exampleshown here a columnar channel located in the center of the bioreactor 1,which is preferably of cylindrical shape. The carriers 7 are stackedalong the same direction. The stacking occurs in one embodiment by meansof mechanical connections defining the side wall of the columnar channel5. These mechanical connections may fix the orientation of each carrierin the stack, but alternatively may leave freedom for independentrotation of each of the carriers. Clearly, it is by no means excludedthat the stack of carriers including the columnar channel could bemanufactured as one piece, for instance by means of a moulding process,and/or that adhesive or mechanical fixtures (screw or the like) are usedfor fixing portion of the stack. However, separate manufacture of thecarriers has the advantage that the stack becomes modular, so as to bemade larger or shorter dependent upon the intended use and neededconditions.

Preferably, as shown in this FIG. 2 , the columnar channel 5 does nothave connections to individual levels 6 extending between adjacentcarriers 7 in the bioreactor 1. This has the advantage that the columnarchannel 5 may be used as a mixing and dissolution vessel. In thisexample, the bioreactor 1 is provided with several ports 13, 14, 15,e.g., a filter 13 for gas exchange, a port 14 for the addition of liquidcomponents, in particularly a solvent, solution, suspension, dispersion,and a port 15 for the addition of gaseous components, for instance air,oxygen or CO₂. Particularly any bubbles 16 resulting from the additionof gaseous components are better prevented from entering the levels 6between the carriers 7. The bioreactor 1 is provided with an impeller 9for stirring the medium. The impeller is typically, and particularly inlaboratory versions of the present bioreactor 1, a magnetic impeller.However, a mechanically driven impeller is not excluded.

This impeller 9 is further responsible for providing the flow of mediumthrough the bioreactor. However, if desired, a separate pump may be usedto control and drive such flow. The impeller 9 may be provided in thelower cavity 3, at the end of the columnar channel 5. It is observedthat the lower cavity 3 may be separated from the columnar channel bymeans of a wall having one or more apertures, i.e., access ports for themedium. Such separation allows that the flow in the columnar channel 5is more vigorous than in the levels 6 of the reactor 1. In such a mannerboth an appropriate mixing and an appropriate flow rate for the cellsmay be achieved.

The bioreactor 1 of this embodiment is provided with at least one inletport 21 at its first side. This inlet port 21 is primarily intended forfilling and emptying of the reactor. However, it is by no means excludedthat this inlet port 21 forms the port to an external circulation systemthat is used in addition to the internal circulation system. In suchcase, typically, at least one outlet port is present on the second side12 of the reactor. If desired, the inlet port 21 and such outlet port 12may be reversed.

As shown in FIG. 2 , the inlet ports 13-15 are suitably present in theupper cavity 4. This cavity 4 further leaves space for sensors 23. Itwill be understood by the skilled person that the bioreactor 1 is at itssecond side 12 preferably closed so as to maintain physical conditionsin the best probable manner.

FIGS. 3 to 7 show a plurality of diagrammatical top views of thedifferent embodiments of the carrier 7 in accordance with theinventions. All these embodiments show circular carriers 7 with openspaces 2 that are provided along several lines from the center 17 of thecarrier 7 to its edge 18 so as to include rotational symmetry. It ishowever by no means excluded that the carriers 7 may have another shape.It is moreover not excluded that the open spaces 2 are oriented alongcircles at around the center 17 rather than on radial lines. The carriercomprises open spaces 2 and solid carriers 27.

FIGS. 3 and 5 show embodiments based on hole-shaped open spaces 2. FIGS.4, 6 and 7 show embodiments based on groove-shaped open spaces 2,wherein grooves extend substantially from the center 17 to the side edge18. Though not shown, the groove-shaped open spaces and the hole-shapedopen spaces may be combined into a single carrier 7 design. Though notshown, the groove-shaped open spaces may be subdivided into a series oftrench-shaped open spaces and the hole-shaped open spaces may be widenedto get such trench-shaped open spaces.

FIGS. 3 and 4 show embodiments in which the surface area of the openspace 2 is independent of the distance to the center 17. FIGS. 5 and 6show embodiments in which the overall surface area covered by the openspaces 2 in at least one of the carrier 7 increases faster withincreasing radial distance to the geometrical center of the carrier 7.FIG. 6 shows a preferred embodiment in which the surface area of theopen spaces 2 increases with the distance to the center 17.

FIG. 5 shows thereof an implementation in which the density of openspaces 2, each of uniform size, increases with increasing radialdistance to the center, e.g., by reduction of the spacing betweenindividual open spaces 2.

FIG. 6 shows an implementation in which the width of the open spaces 2increases with the distance to the center 17.

FIG. 7 shows a specific embodiment, in which the fluid interconnects 2are defined so as to follow rotational movement of the medium in thebioreactor 1, which rotational movement is generated by a pumping system

FIGS. 8 a-8 c demonstrate the flow in the bioreactor in accordance withone embodiment of the invention. For ease of representation, animplementation is shown here, in which the open spaces 2 in a firstcarrier 7 are laterally, e.g., rotationally displaced with respect tothe open spaces 2 in an adjacent second carrier 7. FIG. 8 a hereindiscloses the flow on a microscale, while FIG. 8 c discloses the flow ona macroscale. FIG. 8 b illustrates the microscale in further detail. Itwill be clear that even though the flow on macroscale is along theprincipal direction P of the bioreactor 1, it includes on microscale amajor component 10 extending laterally. FIG. 8 b shows hereof a detailedview clarifying that effectively the flow is primarily lateral insteadof primarily vertical. This is achieved through design, e.g., design ofthe width of the level 6, the size and density of the open spaces 2.

FIGS. 9 and 10 is a cross-sectional diagrammatical view of otherembodiments of the bioreactor 1. The bioreactors 1 of these third andfourth embodiments are quite similar so that they will be discussedtogether. In these embodiments, the stack of carriers 7 is of conicalshape, i.e., each of the carriers comprises at least a portion thatincludes a non-perpendicular (i.e., oblique) angle to the principaldirection of the bioreactor. One advantage of such conical shape is thatit simplifies bubble elimination between individual carriers.Furthermore, emptying and harvesting of the reactor is improved as therisk of forming puddles when emptying the reactor is avoided. Whiletypically only one side of a carrier 7 is used for cell adhesion, it isnot impossible that both sides of the carrier 7 are used for celladhesion. One implementation thereof is the use of an impeller both inthe upper cavity and the lower cavity (see FIG. 14 ). The orientation ofthe bioreactor may then be reversed. This allows that in a firstoperation cells are inserted and are allowed time to settle on a firstcarrier. Thereafter, the reactor orientation is reversed, and furthercells are inserted (if needed) are allowed time to settle on the secondcarrier.

FIG. 13 a-g shows a procedure for using both sides of each carrier in abioreactor according to embodiments of the disclosure. In a first step(FIG. 13 a ), cells C in a medium M are introduced in the bioreactor 1and the cells C are distributed homogeneously via operation of theimpeller 9. In a second step (FIG. 13 b), the cells C are allowed tosettle on a first side of the carriers 7. The arrows stemming from thecells C show the direction of settlement. This is triggered by gravity.In a third step (FIG. 13 c), the medium M is removed from the bioreactor1. In a fourth step (FIG. 13 d), further cells C in a medium M areintroduced in the bioreactor 1 and the cells C are distributedhomogeneously via operation of the impeller 9. In a fifth step (FIG. 13e), the bioreactor 1 is turned upside down after having switched off theimpeller 9. In a sixth step (FIG. 13 f), the cells C are allowed tosettle on a second side of the carriers 7. In a seventh step (FIG. 13g), the bioreactor 1 is turned back in its initial orientation and thecells C can now grow on both sides of each carriers 7.

FIG. 14 shows a bioreactor as in FIG. 2 , wherein a second circulationmeans such as a pump is present in the upper cavity of the bioreactor.This reactor can operate upside down.

FIG. 15 shows a carrier for use in a bioreactor according to embodimentsof the disclosure in which the width of the open spaces 2 in the carrier7 increases with the distance to the center 17. The carrier 7 is herecomposed of alternating solid carriers 27 and open spaces 2 separatinglaterally the solid carriers 27.

FIG. 16 shows a portion of a carrier for use in a bioreactor accordingto embodiments of the disclosure in which open spaces 2 are bridged byridges 19 thereby defining groove-shaped trenches 20.

FIG. 17 shows a diagrammatical top view of a carrier according to adesign for use in a bioreactor according to an embodiment of thedisclosure (left); it also shows a graph showing that the overallsurface area A covered by the open space in the carrier increases fasterwith increasing radial distance D to the geometrical center of thecarrier (right).

FIG. 18 shows a cross-sectional view of the edge of a stack of carriers7 for use in an embodiment of a reactor according to the disclosure.Visible are ridges 10 determining the inter-distance between twoadjacent carriers 7 and the outward projecting ridges 19 that are meantto be embedded in a polymer material.

FIG. 19 shows a perspective view of a bioreactor 1 for the culture ofcells, comprising a stack of carrier 7 defining levels 6. The carriers 7are of two alternative kinds. A first kind is hold in place by its edgeand has a central open space. A second kind is hold in place by beingattached to a central axis 24 and assures an open space for fluidlyinterconnecting two adjacent levels by not extending to the wall closingthe stack.

FIGS. 20-25 relate to an embodiment in which a cell culture device, suchas bioreactor 1, includes carriers 7 _(a) . . . 7 _(n), and is furtheradapted for viewing the growth of the cells C on one or more of theinner carriers from an exterior vantage point. In one possible approach,this may be achieved by providing an optically transparent line of sightfrom the external vantage point V to carrier 7 _(n) through carriers 7_(a) . . . 7 _(n−1). In one particular embodiment, as shown in FIG. 20 ,this may be achieved by providing a surface area A on each carrier 7_(a) . . . 7 _(n−1) on which cells do not grow. This area may beachieved using a chemical treatment, such as by using a process to makethe area hydrophobic to prevent cell adherence or growth, or instead byusing a material that naturally retards or prevents cell adherence andadapting it for growth in areas besides area A (such as by usinghydrophilization). These areas A among the carriers 7 _(a) . . . 7_(n−1) generally align, such that a substantially unobstructed line ofsight is provided to a cell growth area A_(cg) on carrier 7 _(n). Thus,by using a microscope 0 or like device, the cells on this growth areaA_(cg) may easily be observed without interference from cell growth oncarriers 7 _(a) . . . 7 _(n−1). The area A may thus be considered as awindow.

FIG. 21 shows an alternative embodiment, in which the desired line ofsight is provided by providing an area A where cell growth is preventedby using an optically transparent material T associated with eachcarrier 7 _(a) . . . 7 _(n−1), but not the carrier 7 _(n) for which thecell growth observation is desired. Preferably, this material completelyfills the space between adjacent carriers, and thus provides asubstantially continuous optical path from the desired vantage point V.As with the previous embodiment, multiple lines of sight may be providedto provide observations at different levels of the bioreactor. In bothembodiments, it is preferred that the area A is as small as possible toavoid minimizing the cell growth area while still permitting the desiredobservation to be made.

With reference to FIG. 22 , it should be appreciated that differentlines of sight may be provided for different carriers in the samedevice, such as bioreactor 1. Thus, for example, to view the cell growthon carrier 7 _(n), the arrangement is as shown in FIG. 20 , with areasA₁ on which cell growth is substantially prevented. For layer 7 _(n−2),a different optical path is provided by similar areas A₂ on thecorresponding carriers 7 _(a) . . . 7 _(n−3). Furthermore, the path maybe extended in a different area A₃ to reach the growth area of carrier 7_(n+3). As should be appreciated, this pattern may be repeated asnecessary or desired to permit observation on one or more of thecarriers.

While it is possible to use these approaches to viewing the cell growtharea in the cell culturing apparatus, such as bioreactor 1 with stackedcarriers 7 within the same housing, as described herein, it also mayfind use in other applications. Thus, for example, FIGS. 23-25illustrate the use of the embodiments described above in a cell culturedevice (which may comprise a bioreactor 1) comprising a plurality ofstackable carriers 7 _(a), 7 _(b), and 7 _(c), each having a separateinlet. In FIGS. 22-24 , carriers 7 _(a) and 7 _(b) include areas A forpreventing cell growth, which allows for the external observationthrough these areas to the growth area A_(cg) on carrier 7 _(c).Similarly, in FIG. 25 , the optically transparent material T positionedin “cube” style carriers 7 _(a) and 7 _(b) form areas A for preventingcell growth, which allows for the external observation through theseareas to the growth area A_(cg) on carrier 7 _(c) (which of courserequires that any intervening portions of the carriers 7 _(a) and 7 _(b)are optically transparent to a sufficient degree to permit viewing inthe desired manner). In either case, the ability to view lower layersavoids the costly and time-consuming need for having to unstack anyupper carrier(s) in order to view the cell growth for the lowercarrier(s).

FIG. 26 shows an embodiment where the carriers 7 _(a) . . . 7 _(n)include aligned openings to allow for the viewing of the cells onselected carriers. Specifically, the bioreactor 1 may include an inlet21 and an outlet 22 positioned within a housing that contains aplurality of carriers arranged in a stacked configuration. A firstcarrier 7 _(a) may be arranged so as to provide at least one open space2 for allowing a direct view through the first side 12 of the housing(which is at least partially transparent for this purpose) to a growtharea A_(cg) on the next-adjacent carrier 7 _(b). In like manner, thefirst and second carriers 7 _(a) and 7 _(b) may provide aligned openspaces 2 for providing a substantially continuous optical path to thenext-adjacent carrier 7 _(c). This pattern may be repeated as necessaryor desired to allow for the viewing of the growth areas on selectedcarriers from an external vantage point, with the first carrierpreferably having a number of openings corresponding to the innermostcarrier to be viewed, and each successive carrier providing one feweropening than the preceding one. Additionally, it is possible to combineor adapt this approach to allow for the viewing of carriers from thesecond side 11, as indicated by open space 2 in carrier 7 _(n)(provided, of course, the housing is adapted for this purpose).

As should be appreciated, the open spaces 2 may also be arranged forensuring the most desirable flow of fluid, oxygen, and nutrients to thelayers, as outlined above. For instance, there may be two different setsof openings in the carriers (7), such as a first set devoted to“zig-zag” flow for cell nutrition, and a second set of aligned openingsfor observation (and, most preferably, arranged such that the “straight”flow through the aligned openings create is low relative to the mainflow through the offset openings).

As used herein and unless provided otherwise, the term “comprising” isnot synonym of the term “consisting” which has a narrower meaning. Theterm “comprising” should not be interpreted as being restricted to themeans listed thereafter; it does not exclude other elements or steps. Itis thus to be interpreted as specifying the presence of the statedfeatures, integers, steps or components as referred to, but does notpreclude the presence or addition of one or more other features,integers, steps or components, or groups thereof. Thus, the scope of theexpression “a device comprising means A and B” should not be limited todevices consisting only of components A and B. It means that withrespect to the disclosure, the only relevant components of the deviceare A and B. Moreover, the term “comprising” always includes the term“consisting” and when the term “comprising” appears in an embodiment ofthe disclosure, this same embodiment wherein the term “consisting”replaces the term “comprising” is always also an embodiment of thedisclosure.

Furthermore, the terms “first”, “second”, “third” and the like in thedescription and in the claims, are used for distinguishing betweensimilar elements and not necessarily for describing a sequence, eithertemporally, spatially, in ranking or in any other manner. It is to beunderstood that the terms so used are interchangeable under appropriatecircumstances and that the embodiments of the invention described hereinare capable of operation in other sequences than described orillustrated herein.

Moreover, the terms “top”, “bottom”, “over”, “under” and the like in thedescription and the claims are used for descriptive purposes and notnecessarily for describing relative positions. It is to be understoodthat the terms so used are interchangeable under appropriatecircumstances and that the embodiments of the invention described hereinare capable of operation in other orientations than described orillustrated herein.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the disclosure. Thus, appearances of the phrases “in oneembodiment” or “in an embodiment” in various places throughout thisspecification are not necessarily all referring to the same embodiment,but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to one of ordinary skill in the art from this disclosure, inone or more embodiments.

As used herein and unless provided otherwise, the term “length” relatesto the longest dimension of an object (e.g., an open space).

As used herein and unless provided otherwise, the term “width” relatesto the largest dimension of an object taken at right angle to itslength. The “width” is therefore never longer than the “length”.

The foregoing descriptions of various embodiments of the invention areprovided for purposes of illustration, and are not intended to beexhaustive or limiting. Modifications or variations are also possible inlight of the above teachings. The embodiments described above werechosen to provide the best application to thereby enable one of ordinaryskill in the art to utilize the disclosed inventions in variousembodiments and with various modifications as are suited to theparticular use contemplated. All such modifications and variations arewithin the scope of the invention.

1.-122. (canceled)
 123. A bioreactor for use in culturing cells,comprising: a plurality of carriers, each of the plurality of carriershaving a first area adapted for cell adherence, and at least one of theplurality of carriers having a second area adapted for preventing celladherence; and a piece of optically transparent material associated witheach of the at least one of the plurality of carriers having the secondarea, the piece of optically transparent material positioned in a spacebetween adjacent carriers such that the space is completely filled toprovide a substantially continuous optical path from an external vantagepoint through the plurality of carriers to a desired area of cell growthin one of the plurality of carriers.
 124. The bioreactor according toclaim 123, further comprising a second piece of optically transparentmaterial associated with each of the at least one of the plurality ofcarriers having the second area, the piece of optically transparentmaterial positioned in a space between adjacent carriers such that thespace is completely filled to provide a second substantially continuousoptical path is created to a second desired area of cell growth in oneof the plurality of carriers.
 125. The bioreactor according to claim123, wherein the second area adapted for preventing cell adherence ishydrophobic.
 126. The bioreactor according to claim 123, furtherincluding a single housing for receiving the plurality of carriers. 127.The bioreactor according to claim 123, wherein the plurality of carriersinclude stackable trays or cubes.
 128. The bioreactor according to claim123, wherein the second area for preventing cell adherence constitutes awindow.
 129. The bioreactor according to claim 123, wherein each of theplurality of carriers includes a separate inlet.
 130. The bioreactoraccording to claim 123, wherein each of the plurality of carriersincludes open spaces configured to ensure the most desirable flow offluid, oxygen, and nutrients to layers of cells formed on the pluralityof carriers.
 131. A bioreactor comprising a stack of carriers and anoptically transparent solid material positioned in a space betweenadjacent carriers and completely filling said space such that anoptically transparent path is created from a vantage point external tothe bioreactor through at least one carrier to one or more innercarriers.
 132. The bioreactor according to claim 131, wherein a cellgrowth area is on a surface of the one or more inner carriers.
 133. Thebioreactor according to claim 132, wherein the optically transparentsolid material is associated with each of the at least one carrierstacked on top of the one or more inner carriers, but not the one ormore inner carriers.
 134. The bioreactor according to claim 133, whereinthe optically transparent solid material prevents cell growth on an areaof each of the at least one carrier for which said material isassociated.
 135. The bioreactor according to claim 131, wherein thestack is vertical.
 136. A method for viewing growth of cells in a cellculture device from an external vantage point, comprising: providing astack of carriers C_(a) . . . C_(n) forming the cell culture device;generating a surface area on each carrier C_(a) . . . C_(n−1) whereincells do not grow; creating an optically transparent line of sight fromthe external vantage point to carrier C_(n) through carriers C_(a) . . .C_(n−1); and viewing an area of cell growth on carrier C_(n).
 137. Themethod according to claim 136, wherein the cell culture device is abioreactor.
 138. The method according to claim 136, wherein the viewingstep includes utilizing a microscope.
 139. The method according to claim136, wherein the generating step includes utilizing a chemical treatmentto prevent cell adherence or growth on the surface area of each carrierC_(a) . . . C_(n−1).
 140. The method according to claim 136, wherein thegenerating step includes utilizing an optically transparent materialassociated with each carrier C_(a) . . . C_(n−1) but not carrier C_(n).141. The method according to claim 140, wherein the utilizing stepincludes completely filling a space between adjacent carriers with theoptically transparent material.
 142. The method according to claim 136,further including the steps of: generating a second surface area on eachcarrier C_(a) . . . C_(n−3) wherein cells do not grow; creating a secondoptically transparent line of sight from another external vantage pointto carrier C_(n−2) through carriers C_(a) . . . C_(n−3); and viewing asecond area of cell growth on carrier C_(n−2.)